Composition of rapamycin, dimethylsulphoxide and water for use in the treatment of fatty liver disease

Abstract

The present invention relates to a composition comprising rapamycin, dimethylsulfoxide and water for use in the treatment of fatty liver of a liver graft. The invention also relates to a composition comprising rapamycin, dimethylsulfoxide and water obtainable by a preparation method and a method of administration. The invention also relates to the use of a composition comprising rapamycin, dimethylsulfoxide and water for treating fatty liver of a liver graft.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a composition comprising rapamycin, dimethyl sulfoxide and water for use in the treatment of steatosis of a liver graft. The invention also relates to a method of preparation and a method of administration of said composition as well as its use for treating steatosis of a liver graft. TECHNOLOGICAL BACKGROUND

[0002] Liver transplantation (LT) remains the only curative treatment for end-stage cirrhosis and for some early-stage primary liver cancers. The main indications are hepatocellular carcinoma and decompensated cirrhosis. For several years, the number of liver transplants has continued to increase, with long-term results constantly improving. However, the organ shortage remains a significant limitation to liver transplantation and is the cause of significant mortality on the waiting list, estimated at between 10% and 20%. In 2 years, data report no decrease in the incidence of deaths per 1,000 patients per year despite a 5.6% increase in transplants (Agence de la Biomédecine).

[0003] Faced with this organ shortage, teams are increasingly taking livers from elderly donors and / or those with "extended criteria."

[0004] In France, steatosis is the leading cause of liver graft rejection. In 2021, out of 336 livers offered but not harvested, nearly 72 grafts were rejected due to their steatotic appearance (i.e., lipid accumulation in the hepatocytes). Similarly, out of 34 livers harvested but not transplanted, half were rejected due to their steatotic appearance.

[0005] In practice, grafts with steatosis of more than 30% are considered non-transplantable. Indeed, these grafts are more fragile and present an increased risk of dysfunction due to a very low ischemia-reperfusion tolerance threshold.

[0006] However, the use of this type of steatotic graft would allow 10% more patients to be transplanted each year. Furthermore, given the constant increase in the prevalence of obesity (15% to 35% of the Western population), the number of steatotic grafts is expected to continue to grow in the coming years.

[0007] Innovative approaches are currently being explored to increase the number and improve the quality of these grafts. These methods aim to reduce the gap between the number of recipients and the available organ pool. One such approach is dynamic preservation (DP), which is a procedure in which organs are perfused ex vivo after their collection. The objectives of PM are multiple and include the mitigation of ischemia-reperfusion (IR) injury, the ability to extend the preservation time ex live,improving organ condition and function, as well as assessing organ viability prior to transplantation.

[0008] Currently, there are no validated preservation techniques or pharmacological treatments to treat liver graft steatosis.

[0009] There is therefore a need for treatments for liver graft steatosis. Indeed, any therapeutic solution that would allow the use of liver grafts initially deemed untransplantable would be a major step forward in increasing the pool of available organs and thus reducing mortality among patients on the waiting list. SUMMARY OF THE INVENTION

[0010] A first subject of the present invention relates to a composition comprising rapamycin, dimethyl sulfoxide and water for use in the treatment of steatosis of a liver graft.

[0011] It has been particularly highlighted that the administration of such an ex composition vivo to the liver graft, for example by perfusion in normothermic conditions, could make it possible to treat steatosis, greater than 30%, of a liver graft initially deemed non-transplantable.

[0012] The invention also relates to said composition capable of being obtained by a preparation process comprising the following steps: 1) Dissolving rapamycin with dimethyl sulfoxide at a temperature above 18°C; 2) Adding water at a temperature above 18°C ​​to the solution of rapamycin and dimethyl sulfoxide obtained in step 1; 3) Sterilizing the solution obtained in step 2, preferably by filtration.

[0013] The invention also relates to a method of administering said composition, comprising the following steps: 1) Installation of a liver graft on a perfusion machine in hypothermic condition, then in normothermic condition; 2) Injection into the perfusion circuit of the composition comprising rapamycin, dimethyl sulfoxide and water.

[0014] The invention also relates to the use of a composition comprising rapamycin, dimethyl sulfoxide and water for treating steatosis of a liver graft. DETAILED DESCRIPTION

[0015] A first subject of the present invention relates to a composition comprising rapamycin, dimethyl sulfoxide and water for use in the treatment of steatosis of a liver graft.

[0016] In the context of the present invention, the terms "treat", "treatment" etc. indicate an improvement in the percentage of steatosis on the liver graft.

[0017] Steatosis, commonly known as "fatty liver disease," is the accumulation of fat in the liver. Steatosis is directly linked to overweight and obesity.

[0018] Preferably, the steatosis of the liver graft is greater than 30%. The degree of steatosis allows the grafts to be classified into three groups: Less than 30%: transplantable graft, Between 30% and 60%: potentially transplantable graft, after treatment (infusion, pharmacological treatment) and viability assessment; More than 60%: non-transplantable graft.

[0019] Rapamycin, also known as sirolimus, was initially discovered as an antifungal metabolite produced by Streptomyces hygroscopicus from a soil sample from Easter Island. The immunosuppressive and antiproliferative properties of this molecule against mammalian cells have generated interest in identifying its mechanism of action (Li J et al., 2014).

[0020] It is an mTOR (mechanistic target of rapamycin) inhibitor that targets the mTORC1 protein kinase and inactivates this signaling pathway.

[0021] The mTOR signaling pathway is complex and the consequences of this inhibition on cellular functions are multiple, including two major actions: Role in hepatic lipid metabolism via defatting properties, including lipogenesis, triglyceride homeostasis, and autophagy. Rapamycin is used to prevent organ rejection and is associated with increased serum cholesterol (hypercholesterolemia) and triglycerides (hypertriglyceridemia), which are monitored biologically. Immunological action: the function and production of immune cells. This role is particularly important in transplantation because inhibition of the mTOR / FKBP-12 complex blocks the proliferation of T and B lymphocytes in response to IL-2 and IL-15, which helps limit allograft rejection (clinical use in the prophylaxis of graft rejection).

[0022] Rapamycin corresponds to the active ingredient of the composition according to the invention.

[0023] Dimethyl sulfoxide or DMSO is an organosulfur compound and a polar aprotic solvent. This compound is a solubilizing agent.

[0024] Preferably, the composition according to the invention is in the form of an injectable solution.

[0025] By “solution” is meant a liquid composition free from visible particles, using the procedure according to the European and / or American Pharmacopoeia.

[0026] “Injectable solution” means a solution that meets the requirements of the European and / or American Pharmacopoeias and is sufficiently liquid to be injected. in vivo or ex live, preferably in an infusion circuit.

[0027] Rapamycin is an unstable product in solution. There are currently no pharmaceutically acceptable products containing rapamycin in solution. This form is essential for injection in a liver conditioning context for transplantation.

[0028] The composition according to the invention is intended in particular to be used in the treatment of steatosis of a liver graft in an operating room (at room temperature). However, dimethyl sulfoxide has a melting point of 18.5°C which can cause problems of use, particularly in the operating room because the product would be in the solid state (room temperature being close to the melting point). Furthermore, water has a melting point of 0°C. Advantageously, the mixture of dimethyl sulfoxide and water allows a lowering of the freezing point of the composition. Indeed, the melting point of the composition is lower than -18.5°C, thus making it possible to obtain a composition having improved physicochemical stability compared to a composition comprising only DMSO. Thus, the composition according to the present invention is stable during storage and use.

[0029] This composition allows the stabilization of rapamycin in the form of a ready-to-use solution. Thus, the injection of the rapamycin composition in the form of solution can be carried out at the time deemed most appropriate by specialized transplant teams.

[0030] In a first embodiment, the composition according to the invention is used in ex vivo administration to the liver graft, in particular during perfusion on a machine.

[0031] Several types of protocols for the preservation of liver grafts before transplantation and ex perfusion vivohave been proposed, namely hypothermic oxygenated perfusion or HOPE (4°C), continuous normothermic preservation or cNMP (37°C) and sub-normothermic perfusion (21°C) (Muller X et al., 2022). In practice, the most therapeutically effective perfusion mode, giving the best results, consists of combining several conditions starting with a perfusion in hypothermic conditions followed by progressive warming up to normothermia (Boteon YL et al., December 2018). The first phase in oxygenated hypothermia allows the reconstitution of Adenosine TriphosPhate (ATP) stores with an oxygen supply in conditions of reduced metabolism while the second in normothermia at 37°C allows the maintenance of the organ in physiological conditions and therefore has the advantage of being able to test the viability of the organ and to carry out pharmacological interventions.

[0032] Preferably, the ex administration vivois a perfusion in normothermic conditions.

[0033] A "normothermic condition" means a temperature between 36°C and 38°C.

[0034] Preferably, the administration of the composition according to the invention is carried out as a bolus in an ex perfusion circuit vivo with the continuous circulation of a suitable medium (red blood cells, albumin, nutrient buffer medium for cells such as Krebs solution) in normothermic conditions for 12 hours at a temperature between 36°C and 38°C.

[0035] It should be noted that administration in an ex perfusion circuit vivoeliminates the risk of systemic toxicity, as rapamycin is delivered only to the organ. It also allows doses to be increased without the risk of extrahepatic toxicity. Normothermia ensures normal physiological and metabolic functioning of the liver and thus allows for liver quality checks and specific pharmacological studies of rapamycin (pharmacokinetics and pharmacodynamics).

[0036] Furthermore, the composition according to the invention is effective on liver grafts without altering cell viability.

[0037] Preferably, the concentration of rapamycin in the composition is less than or equal to 1 mg / mL, preferably between 0.4 mg / mL and 1 mg / mL, even more preferably 0.5 mg / mL.

[0038] Preferably, the circulating concentration of rapamycin is between 120 ng / mL and 360 ng / mL, preferably 180 ng / mL.

[0039] Preferably, the composition according to the invention comprises between 0.02% and 0.5% of rapamycin, expressed as a mass percentage.

[0040] Preferably, said composition comprises ultra-pure water, also referred to as "water" in the present invention.

[0041] Ultrapure water is water that has been purified to high specification levels. Typically, the water contains only H 2 0, along with a balanced number of H + < and OH - < ions. It has a resistivity of 18.2 MO.cm, a TOC (Total Organic Carbon) < 10 ppb, and a bacteria count < 10 CFU / ml. To be classified as ultrapure, the water must contain no detectable endotoxins.

[0042] Preferably, the composition according to the invention comprises between 70% and 99% of dimethyl sulfoxide and between 1% and 30% of water, preferably 85% of dimethyl sulfoxide and 15% of water, expressed as a volume percentage.

[0043] Even more preferably, the composition according to the invention comprises 0.5 mg / mL of rapamycin, 85% of dimethyl sulfoxide and 15% of water, expressed as a volume percentage.

[0044] Preferably, dimethyl sulfoxide and water are pharmaceutically acceptable.

[0045] Advantageously, the dimethyl sulfoxide / water ratio is between 2.5 and 95, preferably between 2.6 and 92 and even more preferably between 2.65 and 91.25.

[0046] Preferably, the composition according to the invention is stored between -30°C and -10°C, preferably at -20°C.

[0047] The composition according to the invention can be stored for a period of between 6 and 24 months at a temperature of between -30°C and -10°C.

[0048] In a second embodiment, the composition comprising rapamycin, dimethyl sulfoxide and water as described above is used in an administration in vivoin the donor before the liver graft is harvested.

[0049] Preferably, the administration in vivo is an intravenous administration.

[0050] The present invention also relates to a composition comprising rapamycin, dimethyl sulfoxide and water for use in the treatment of steatosis of a liver graft, obtainable by a preparation process comprising the following steps: 1) Dissolving rapamycin with dimethyl sulfoxide at a temperature above 18°C; 2) Adding water at a temperature above 18°C ​​to the solution of rapamycin and dimethyl sulfoxide obtained in step 1; 3) Sterilizing the solution obtained in step 2, preferably by filtration.

[0051] The preparation process by which the composition according to the invention is capable of being obtained or obtained by said preparation process may comprise a final step in which the solution obtained in step 2 is treated with an inert gas such as nitrogen, argon, carbon dioxide, xenon or krypton. The purpose of this final step is to limit the risk of oxidative degradation of rapamycin.

[0052] Preferably, in step 1 and step 2, the temperature is greater than 18°C ​​and less than 50°C, preferably between 19°C and 30°C, more preferably between 20°C and 25°C, and in particular, the temperature is 20°C.

[0053] Preferably, between step 2 and step 3, a temperature above 18°C ​​and below 50°C, preferably between 19°C and 30°C, more preferably between 20°C and 25°C, and in particular, the temperature is 20°C is maintained in order to take into account the exothermicity of the mixture and the expansions / contractions of volumes linked to the mixture itself of the two solvents, DMSO and water, and to the variations in temperature.

[0054] Preferably, the volume of the solution obtained in step 2 can be made up with dimethylsulfoxide at a temperature above 18°C ​​and below 50°C, preferably between 19°C and 30°C, more preferably between 20°C and 25°C, and in particular, the temperature is 20°C. In particular, the amount of dimethylsulfoxide added makes it possible to obtain a composition comprising between 70% and 99% of dimethylsulfoxide, expressed as a volume percentage.

[0055] Preferably, rapamycin is dissolved in step 1 at a rate of 5 mg / mL.

[0056] Preferably, sterilization step 3 can be carried out by filtration.

[0057] Preferably, the filters used are compatible with the solvents used, without compromising the rapamycin content. The pore size of the filters is preferably between 0.1µm and 0.22µm, even more preferably 0.2µm.

[0058] Preferably, the filtration is carried out with a nylon or polytetrafluoroethylene filter. The present invention also relates to a method of administering a composition as described above, comprising the following steps: 1) Installation of a liver graft on a perfusion machine in hypothermic condition, then in normothermic condition; 2) Injection into the perfusion circuit of the composition comprising rapamycin, dimethyl sulfoxide and water.

[0059] A "hypothermic condition" means a temperature between 4°C and 8°C.

[0060] Preferably, in step 1, the hypothermic condition is carried out at a temperature between 4°C and 8°C for 1 hour to 2 hours. Preferably, in step 1, the normothermic condition is carried out at a temperature between 36°C and 38°C for 12 hours to 120 hours.

[0061] Preferably, between the hypothermic condition and the normothermic condition of step 1, an intermediate phase of warming in sub-normothermic condition, at a temperature between 18°C ​​and 22°C, preferably at a temperature of 21°C is implemented for 20 minutes to 1 hour, preferably for 30 minutes.

[0062] Preferably, in step 2, the injection into the perfusion circuit of the composition of rapamycin, dimethyl sulfoxide and water is carried out 2 hours after the start of the normothermic condition.

[0063] The present invention also relates to the use of a composition comprising rapamycin, dimethyl sulfoxide and water for treating steatosis of a liver graft.

[0064] The use according to the present invention advantageously comprises a composition comprising rapamycin, dimethyl sulfoxide and water administered ex vivo to the liver graft and advantageously the concentration of rapamycin in the composition is less than or equal to 1 mg / mL, preferably between 0.4 mg / mL and 1 mg / mL. Advantageously, the circulating concentration of rapamycin is between 120 ng / mL and 360 ng / mL, preferably 180 ng / mL.

[0065] Advantageously, the composition comprises between 70% and 99% of dimethyl sulfoxide and between 1% and 30% of water, preferably 85% of dimethyl sulfoxide and 15% of water, expressed as a volume percentage.

[0066] All the general and specific aspects described above for the composition comprising rapamycin, dimethyl sulfoxide and water also apply to its use.

[0067] Other characteristics and advantages of the invention will appear in the following examples, given for illustrative purposes, with reference to: EXAMPLES Example 1: Development of a 0.5 mg / mL rapamycin solution for ex-injection vivo of a liver graft 1. Materials and Methods

[0068] Table 1: Qualitative and quantitative composition of the composition of example 1. Composition Quantity Function Reference standards Manufacturer / Supplier Rapamycin 500mg Active ingredient Manufacturer's Certificate (AMRI) AMRI / INRESA Dimethyl sulfoxide 850mL Solubilizing agent Monograph 0763 (PE 11th edition) 1. Gaylord Pharmaceutical / INRESA 2. Merck / Sigma-Aldrich Chemistry Ultra-pure water Qsp 1000mL - - Millipore Merck Table 2: Primary packaging of the composition of Example 1. Primary packaging Kind Manufacturer / Supplier Packaging Amber glass bottle, type I Schott / ADELPHI Closing device Chlorobutyl 4432 / 50 stopper covered with a Flurotec ® elastomer film West Pharmaceutical / ADELPHI

[0069] This type of primary packaging of the composition was chosen to reduce the risk of container / content interaction and to protect the rapamycin from light. 2. Formulation development

[0070] The galenic prerequisites of the rapamycin solution to be developed are based on: Solubilization and stabilization of rapamycin to achieve an adequate target concentration after dilution to i) be able to handle the product and ii) limit the amount of solvent to be injected. Sterility of the solution and absence of visible recrystallization in a saline environment.

[0071] From a biological point of view, it is expected that there will be no harmful impact on the cellular viability of liver tissues (liver slices allowing the liver structure to be preserved) and hepatocytes from steatotic livers (biocompatibility), no rapid uptake by red blood cells (bioavailability) and effective stimulation of fat removal on liver tissues and then on explanted livers. 2.1 Solubility and choice of solvent

[0072] Several publications report the formulation of rapamycin from liposomes (Rouf MA et al., 2009, Ghanbarzadeh S et al., 2014), cyclodextrins (Dou Y et al., 2016, Rouf MA et al., 2007), micelles (Rapamycin encapsulated in dual-responsive micelles for cancer therapy, 2023), DMSO (commercialization of rapamycin solubilized in DMSO up to 2.5 mg / mL, non-pharmaceutical grade) or by the cosolvent method (Simamora P et al., 2001).

[0073] However, solubilized forms from supramolecular systems, especially liposomes and micelles, have been excluded due to their complexity and their ability to modify the distribution of an active ingredient. The aim of the present invention is to make rapamycin immediately available for the liver graft to be treated on a perfusion circuit.

[0074] DMSO is a pharmaceutical excipient for which rapamycin has a high solubility, greater than 250 mg / mL (Simamora P et al., 2001).

[0075] DMSO is used for intravesical administration as a 50 / 50 DMSO / H 2 O aqueous solution (RIMSO-50 ®< , solution for intravesical instillation indicated for the treatment of interstitial cystitis) under compassionate use authorization and is approved by the Food and Drug Administration (FDA). 2.1.1 Solubility study

[0076] A solubility study was carried out on DMSO / H2O mixtures, from 40% to 100% (v / v) in DMSO and rapamycin concentrations between 0.4 and 4 mg / mL.

[0077] The evaluation of rapamycin solubilization based on different DMSO concentrations is presented in Table 3 below: Table 3: Evaluation of rapamycin solubilization as a function of different DMSO concentrations. DMSO / H2O (volume %) [Rapamycin] (mg / mL) Solubility at room temperature 40 0,4 No (milky appearance with crystals) 1 No (milky appearance with crystals) 4 No (milky appearance with crystals) 60 0,4 Yes 1 Yes 4 No (milky appearance with crystals) 80 0,4 Yes 1 Yes 4 Yes 100 0,4 Yes 1 Yes 4 Yes

[0078] The results show that rapamycin is soluble for the studied concentration range 0.4 and 4 mg / mL for DMSO concentrations equal to or greater than 80% as well as for a concentration of 60% in DMSO for rapamycin concentrations equal to or less than 1 mg / mL (Table 3).

[0079] Following these results, a new study was launched on a more restricted range in DMSO, with 3 concentration levels (70%; 85%; 100%) and the same 3 levels of rapamycin (0.4; 1; 4 mg / mL) (Table 4). Table 4: Evaluation of rapamycin solubilization in DMSO concentrations of 70%, 85% and 100%. DMSO / H2O (volume %) [Rapamycin] (mg / mL) Solubility at room temperature 100 4 Yes 1 Yes 0,4 Yes 85 4 Yes 1 Yes 0,4 Yes 70 4 Yes 1 Yes 0,4 Yes

[0080] The results show that rapamycin up to 4 mg / mL is soluble at DMSO concentrations of 70% to 100%, expressed as volume percentage. 2.1.2 Precipitation risk assessment

[0081] An assessment of the risk of precipitation of formulations (70%; 85%; 100% in DMSO) in the blood was carried out under unfavorable conditions, commonly referred to in English as “worst case”.

[0082] A worst-case condition is defined as a condition or set of conditions that, compared to ideal conditions, are unfavorable and carry the greatest risk of product or process failure. This includes the circumstances and process limits (upper and lower), within the limits of operating procedures.

[0083] The worst case conditions for precipitation risk assessment were defined as follows: 1) low acceptor medium: absence of solvents, cells (red blood cells), proteins, lipids or lipoproteins that can solubilize rapamycin or form a colloidal suspension. Thus, a saline phosphate buffer at pH 7.4°C and 37°C is considered low acceptor. 2) High solute concentration: a rapamycin concentration higher than expected in vivoafter dilution presents a risk of precipitation. Concentrations after dilution of 4 to 40 µg / mL are much higher than those expected in clinical practice after dilution in the perfusion circuit and are therefore considered at risk of precipitation.

[0084] To evaluate this parameter, a 100-fold dilution of the different rapamycin solutions previously presented was carried out in a phosphate-buffered saline maintained at 37°C, devoid of lipid bilayer membrane systems (rapamycin being distributed to the level of cellular elements in the blood).

[0085] The assessment of the risk of precipitation of solutions in the blood via dilution to 100 of the different solutions in a phosphate buffer saline solution at 37°C is presented in Table 5 below: Table 5: Evaluation of the risk of precipitation of solutions in the blood via 100-fold dilution of the different solutions in a phosphate buffer saline solution at 37°C. DMSO / H2O (volume %) [Rapamycin] before dilution (mg / mL) [Rapamycin] after dilution to 100 e< (µg / mL) Visual appearance at room temperature 100 4 40 Disorder* 1 10 Not cloudy** 0,4 4 Not cloudy 85 4 40 Disorder 1 10 Not cloudy 0,4 4 Not cloudy 70 4 40 Disorder 1 10 Not cloudy 0,4 4 Not cloudy * Recrystallization ** Absence of crystals (visual appearance)

[0086] When diluted in phosphate-buffered saline, all 4 mg / mL rapamycin solutions, regardless of the proportion of DMSO, induced cloudiness of the phosphate-buffered saline. It would therefore appear that rapamycin is, at this concentration, suspended in phosphate-buffered saline.

[0087] Solutions equal to or less than 1 mg / mL of rapamycin, regardless of the proportion of DMSO, did not show any impact on the appearance of the stamp. According to all the analyses carried out on DMSO-based rapamycin solutions, it is therefore preferred: A minimum concentration of 70% (v / v) in DMSO to ensure its solubilization (Table 4). A concentration less than or equal to 1 mg / mL of rapamycin to limit the risk of recrystallization after dilution in the perfusion medium (Table 5). 3. Development of the manufacturing process 3.1 Stages of the manufacturing process carried out in the laboratory

[0088] The steps required to manufacture the 0.5 mg / mL rapamycin solution are shown in Table 6 below: Table 6: Manufacturing process carried out in the laboratory. Steps Description Environment Material(s) used Device used 1 Weighing and preparing the mother solution Solubilization of rapamycin at 5 mg / mL: CMR Room Balance - weigh 50mg of rapamycin - 15 mL glass beaker - place them in a 10mL vial - cup - rinse the cup with DMSO - 10mL amber glass vial - vial stopper - carry out the QSP in DMSO - spatula - homogenize by inversion 2 Preparation of the final solution - add 5mL of the previously prepared stock solution into a 50mL flask Under the hood - 15mL glass beakers Not applicable - add 7.5mL of ultrapure water - 50mL glass beaker - add DMSO without reaching the gauge line - combitips advanced ® syringe< 5-10mL - close and wait for it to return to room temperature (approximately 30 minutes) - 50mL amber glass vial - vial stopper - carry out the DMSO QSP - homogenize 3 Filtration - pour the solution into a beaker Under the hood - 50mL glass beakers Manual filtration - draw the solution with a syringe - 60mL plastic syringe - filter through 0.2µm nylon or PTFE into a beaker - 0.2µm nylon or PTFE filter 4 Filling - distribute 2.5 mL of the filtered solution into the primary packaging Under the hood - 2.5 mL syringe Not applicable - Type I amber glass vials - 4mL (Schott) 5 Inerting - inert with nitrogen Under the hood - time: between 5 - 10 seconds Not applicable - pressure (Pressure set on a pressure gauge and a manual flow adjuster, in the absence of a digital indicator 6 Sealing Under the hood - 13mm chlorobutyl coated cap 4432 / 50 FluroTec ®< (Westar) Manual sealing system - stopper the bottles - seal - aluminum capsule 7 Storage - place the samples in a -20°C freezer Under the hood N / A Freezer 3.2 Sterility of the formulation

[0089] The present invention aims to provide a single-dose solution for injection during machine perfusion of the liver graft. The sterility of the solution must comply with the requirements of the European Pharmacopoeia.

[0090] In order to comply with the requirements of the European Pharmacopoeia regarding sterility and to reduce the risks of degradation of rapamycin by heat or irradiation (heat-sensitive nature), sterilizing membrane filtration is the method chosen to reduce contamination by viable or non-viable particles. The manufacturing process therefore includes a sterilizing filtration step. 3.3 Choice of sterilizing filter

[0091] Polytetrafluoroethylene (PTFE), polypropylene (PP) and nylon filters are known to be compatible with DMSO. Sterilizing filtration of the solutions was carried out with sterile 25 mm diameter PTFE and nylon filters (pore size: 0.2 µm) and Sartoscale ® filter with nylon membrane (Sartolon Sartoscale Disposable, pore size 0.45 + 0.2 µm). 3.3.1 PTFE filter

[0092] The tested solutions were subjected to a filtration study on a 0.22 µm PTFE filter (syringe filter, diameter 25 mm) after one week of storage at 4°C. The rapamycin content was evaluated via their areas on the chromatograms before and after filtration during the same analysis sequence. An absence of impact of the rapamycin content after filtration is observed (Table 7). The solutions are filterable on 0.22 µm PTFE filters. Table 7: Evaluation of the impact of filtration on 0.22 µm PTFE filter on rapamycin content. DMSO (volume %) [rapamycin] (mg / mL) Ratio of rapamycin areas before / after filtration (%) 100 4 102% 1 99% 0,4 99% 85 4 100% 1 101% 0,4 100% 70 4 99% 1 100% 0,4 99% 3.3.2 Nylon filter - Nalgene ® Sterilizing Filter<

[0093] The filter selected for carrying out the pre-stability and stability indicator tests is the 0.2 µm sterilizing nylon filter Nalgene ® from the supplier Thermo Scientific.

[0094] Rapamycin levels after filtration are within specifications. - Sartoscale ® Sterilizing Filter<

[0095] The filter selected for testing filter integrity is the 0.2 µm Sartoscale ®< sterilizing filter (nylon membrane, polypropylene body) from the supplier Sartorius.

[0096] The integrity after sterilizing filtration was carried out using the water bubble point test method. The test complies with the specifications under laboratory conditions. 3.4 Determination of conservation and storage conditions - Pre-stability study

[0097] Pre-stability tests of the formulas were set up to allow the selection of the final formula. They were carried out under the following conditions: Three DMSO concentration levels: 70%; 85%; 100% (v / v). Two rapamycin concentration levels: 133 µg / mL and 500 µg / mL. Timescales: T0, T1, T2, T3, T6, T9 and T12 months. Temperatures: +4°C and -20°C.

[0098] The concentration at 133µg / mL is the lowest concentration chosen to detect related substances.

[0099] The 6 formulas tested are represented in Table 8 below: From T0, whatever the storage conditions (+ 4°C and - 20°C), the appearance of impurities above the carryover threshold is observed for formulas A and B. From T0, whatever the storage conditions (+ 4°C and -20°C), the rapamycin dosages are non-compliant (<95%) for formulas E and F. Up to T6 months, formula C is stable in the freezer (-20°C), Up to T9 months, formulas E, F and D are stable in the freezer (-20°C). However, from T0 months, formulas E and F have a rapamycin content below 95%. At T9 months, for storage in the freezer (-20°C), an impurity appears above the carryover threshold for formula C; this impurity does not appear for formula D. At T9 months, formula D stored at -20°C remains stable. In contrast, an impurity appears above the carryover threshold for formula D stored at 4°C.

[0100] It appears that formula D (rapamycin concentration of 500 µg / mL; 85% DMSO: 15% water) stored at -20°C is the most stable. In addition, the mixture remains in liquid form at -20°C for this DMSO:water proportion, which is of interest for rapid use in the operating room. The exothermic DMSO:water mixture did not impact the stability of rapamycin. - Stability study under normal conditions of use

[0101] In order to reproduce the conditions of the tests for performing perfusion of steatotic livers in the operating room and to determine the storage conditions of the formula upon removal from storage at -20°C, stability studies under normal conditions of use were carried out on closed and opened bottles at 6 times (T0, T2h, T5h, T7h, T17h and T24h) at 20°C + / - 5°C.

[0102] Pre-stability and stability studies under normal conditions of use have thus made it possible to determine the storage conditions. The composition is preserved and stored in a freezer, preferably at -20°C. At this temperature, the product remains liquid. Upon removal from the freezer and upon return to room temperature, it is recommended to use the bottles immediately (between 2 and 7 hours). 3.5 Critical parameters of the manufacturing process

[0103] The critical parameters of the manufacturing process have been identified and are shown in Table 9 below: Table 9: Critical parameters of the manufacturing process. Critical parameter Freedom Beach Objective Environment Manufacturing temperature <50°C Rapamycin stability >18°C Solidification of DMSO at 18°C Environment Manufacturing environment light Filters to identify Ensuring the stability of rapamycin Manufacturing Material of equipment used Polymers PTFE - up to 189°C Polyethylene Polypropylene Ethylene propylene gum - up to 48°C Limiting the solubilization of DMSO with many compounds Manufacturing Material of equipment used metals 304-316 stainless steel Very low corrosion level of DMSO Manufacturing Thermostat or system for slowly adding water to the DMSO ΔH Limit the exothermic reaction Manufacturing Filtered Nylon or PTFE Sterilize the product and remove any particulate contaminants Storage Storage of the finished product -20°C Limit the degradation of rapamycin Storage Storage temperature of DMSO >18°C Solidification of DMSO at 18°C Example 2 : Evaluation of the “degreasing” effect of rapamycin

[0104] The term “defatting” effect refers in particular to a reduction in the level of lipids and / or triglycerides in the liver. 1. In vitro results

[0105] Rapamycin was evaluated on hepatocyte cultures and liver tissues (liver slices allowing the liver structure to be preserved). At a concentration of 200nM of rapamycin, a 19% decrease in intracellular triglyceride levels was observed compared to the control in hepatocytes and a 38% decrease in liver tissues. These results, repeated several times, confirmed the proof of concept of the effect of rapamycin on the "defatting" effect. 2. Results on perfusion circuit without organ

[0106] A liver perfusion machine was used with perfusate consisting of human whole blood maintained at 37°C. Rapamycin was injected directly into the perfusion circuit. Samples were taken at regular intervals to measure the molecule. The experiment was performed 4 times, the objective being to validate the theoretical target concentration of 180 ng / mL defined from previous efficacy and toxicity studies.

[0107] The results showed the achievement of an average concentration of rapamycin of 150 + / - 30 ng / mL which validates the first step. 3. Results on perfusion circuit with pig liver

[0108] The same perfusion protocol was used but with an animal liver. This was taken from a pig in cardiac arrest and then preserved in ice. It was then placed on a perfusion machine, initially in hypothermia (4°C) for 2 hours then at 37°C for 12 hours with an intermediate warming phase (sub-normothermia, 21°C). Rapamycin was injected into the circuit 2 hours after the start of the normothermia phase. Nine experiments were carried out. The objective was to have a pharmacokinetic study of rapamycin.

[0109] Rapamycin assays in whole blood and plasma show a peak concentration 5 minutes after injection of rapamycin into the circuit and then a progressive decrease over time. Rapamycin was also measured in bile and was found as early as 1 hour after injection in the blood and increased as the infusion progressed.

[0110] These results therefore show an uptake of rapamycin by the liver and its metabolism with excretion in the bile. 4. Results on perfusion circuit with human liver

[0111] Similarly, the experiment was conducted with two human livers deemed non-transplantable and used for scientific purposes after authorization from the Biomedicine Agency. These two livers presented significant steatosis (greater than 50%). The viability of these two organs was assessed according to criteria validated in clinical practice. The objective was to evaluate the effect of rapamycin on steatosis. To do this, intracellular triglycerides were measured.

[0112] Regarding pharmacokinetics, this was identical to that observed during perfusion of pig livers.

[0113] For steatosis, the intracellular triglyceride level decreased by 39% 9 hours after rapamycin injection for liver No. 1 and by 31% for liver No. 2. Example 3: Evaluation of the toxicity and efficacy of rapamycin solution

[0114] The solubilization and stability tests as detailed in example 1 made it possible to identify two formulas for in vitro tests: DMSO 85%:water 15% up to 1mg / mL in rapamycin (hereinafter “Rapa-85%”); and DMSO 100% up to 1mg / mL in rapamycin (hereinafter “Rapa-100%”).

[0115] The formulas were compared at equivalent concentration of rapamycin during toxicity and cellular efficacy tests on two models: ▪ Model in vitro(2D): Human hepatocytes in primary culture Evaluation of the effect of rapamycin in a Rapa-100% solution; Comparison of the defatting effect of rapamycin in 2 formulas: Rapa-100% and Rapa-85% to confirm the effectiveness of the defatting effect of rapamycin on steatosis developed in vitro. ▪ Ex model vivo (3D): Human Precision-Cut Liver Slices (hPCLS) or liver tissues in primary culture.

[0116] The toxicity of the formulas was assessed by hepatocyte cell viability and their efficacy by monitoring intracellular triglyceride (TG) concentration. The objective of these tests is to select the appropriate formula and dose of rapamycin.

[0117] The in vitro results are shown in Table 10 below: Table 10: Results on in vitro models 20. Tests carried out Efficiency Toxicity Rapamycin concentration tested Model in vitro: Evaluation of the effect of rapamycin in a Rapa-100% solution - Decrease in lipid droplets by 30% in rapamycin-treated hepatocytes compared to steatotic hepatocytes. No overall effect on human hepatocyte viability was observed. 200nM - Decrease in intracellular triglyceride (TG) content by 24% (p<0.05) in treated steatotic hepatocytes compared to steatotic hepatocytes. Model in vitro: Comparison of the defatting effect of rapamycin in 2 formulas: Rapa-100% and Rapa-85%, on hepatocytes from steatotic liver For [Rapa-85%] at 200 nM, 500 nM or 1000 nM: No overall effect on human hepatocyte viability was observed for the [Rapa-85%] and [Rapa-100%] formula at different concentrations. - Decrease in intracellular lipid droplets by 24%, 25% or 28% respectively (p<0.05) compared to treated steatotic hepatocytes. - Decrease in intracellular TG of 19%, 20% or 10% respectively (p<0.05) compared to treated steatotic hepatocytes. 20nM 200 nM 500 nM 1000 nM For [Rapa-100%] at 200 nM: decrease in lipid droplets and intracellular TG by 19% and 20% compared to vehicle-treated cells.

[0118] The results show that the addition of rapamycin has no impact on hepatocyte viability.

[0119] Furthermore, the results show that the defatting effect of rapamycin on hepatocytes from steatotic liver at several concentrations does not alter cell viability.

[0120] The results in vitro show that [Rapa-100%] or [Rapa-85%] solutions significantly decrease the level of lipid droplets and intracellular TG of steatotic hepatocytes, at different rapamycin concentrations. They also confirmed the effectiveness of [Rapa-85%] and [Rapa-100%] in defatting steatotic hepatocytes without altering cell viability.

[0121] The results ex vivo are indicated in Table 11 below: Table 11 vivo : Results on ex 3D models. Tests carried out Efficiency Toxicity Rapamycin concentration tested Model ex vivo (3D) : fabrics liver (Precision liver slices (PCLS)) - Significant decrease in TG content in PCLS by 39% and 38% compared to vehicle (p<0.05), respectively for [Rapa-100] and [Rapa-85%] at a concentration of 200 nM. No overall effect on viability of slices No liver damage was observed for either formula, regardless of the concentration used. - [Rapa-100%] and - [Rapa-85%] 20 nM 200 nM 500 nM - Significant decrease in intracellular TG by 40% in PCLS treated with [Rapa-100%] at 500 nM compared to PCLS treated with [Rapa-85%] at the same dose (p<0.05). 1000 nM

[0122] The results ex vivo show that [Rapa-100%] or [Rapa-85%] solutions significantly decrease the intracellular TG level at the target concentration in the blood of 200 nM. They also confirmed the effectiveness of [Rapa-85%] and [Rapa-100%] in defatting steatotic PCLS without altering cell viability.

[0123] The liquid form at -20°C of the [Rapa-85%] solution (interest for rapid use in the operating room) and the pre-stability results described previously made it possible to select this formula. Example 4: Compositions comprising rapamycin, dimethyl sulfoxide and water according to the present invention

[0124] Table 12 below represents different formulations of the composition according to the present invention: Painting 12: Different formulations of the composition according to the present invention. Rapamycin concentration (µg / mL) 400 400 400 500 500 500 4000 4000 4000 DMSO volume percentage (%) 70 85 99 70 85 99 70 85 99 Mass percentage of rapamycin (%) 0,04 0,04 0,04 0,05 0,04 0,05 0,37 0,37 0,36 DMSO mass percentage (%) 72,60 86,45 98,87 72,60 86,44 98,87 72,36 86,16 98,56 Mass percentage Water (%) 27,36 13,51 1,09 27,35 13,52 1,08 27,27 13,47 1,08 DMSO / Water ratio (m / m) 2,65 6,40 91,25 2,65 6,40 91,25 2,65 6,40 91,25 REFERENCES

[0125] Li J, Kim SG, Blenis J. Rapamycin: one drug, many effects. Cell Metab. 4 mars 2014; 19(3):373-9. Muller X, Rossignol G, Mohkam K, Mabrut JY. Stratégies de conservation des greffons en transplantation hépatique - progrès et perspectives en France. J Chir Viscérale. 1 oct 2022;159(5):412-22. Boteon YL, Wallace L, Boteon APCS, Mirza DF, Mergental H, Bhogal RH, et al. An effective protocol for pharmacological defatting of primary human hépatocytes which is nontoxic to cholangiocytes or intrahepatic endothélial cells. Vinciguerra M, éditeur. PLOS ONE. 25 juill 2018;13(7):e0201419. Rouf MA, Vural I, Renoir JM, Hincal AA. Development and characterization of liposomal formulations for rapamycin delivery and investigation of their antiproliferative effect on MCF7 cells. J Liposome Res. 2009;19(4):322-31. Ghanbarzadeh S, Khorrami A, Mohamed Khosroshahi L, Arami S. Fusogenic pH sensitive liposomal formulation for rapamycin: improvement of antiproliferative effect. Pharm Biol.juill 2014;52(7):848-54. Dou Y, Guo J, Chen Y, Han S, Xu X, Shi Q, et al. Sustained delivery by a cyclodextrin material-based nanocarrier potentiates antiatherosclerotic activity of rapamycin via selectively inhibiting mTORC1 in mice. J Control Release Off J Control Release Soc. 10 août 2016;235:48-62. Rouf MA, Bilensoy E, Vural I, Hincal A. Abdur Rouf M, Vural I, Bilensoy E, Hincal A, Erol DD. Rapamycin-cyclodextrin complexation: improved solubility and dissolution rate. J Incl Phenom Macrocycl Chem. 1 juin 2011;70(1):167 75. Eur J Pharm Sci - EUR J PHARM SCI. 1 sept 2007;32. Rapamycin encapsulated in dual-responsive micelles for cancer therapy - ScienceDirect [Internet]. [cité 13 févr 2023]. Disponible sur: https: / / www.sciencedirect.com / science / article / pii / S0142961212011556 Simamora P, Alvarez JM, Yalkowsky SH. Solubilization of rapamycin. Int J Pharm. 1 févr 2001;213(1-2):25-9.

Claims

1. A composition comprising rapamycin, dimethyl sulfoxide and water for use in the treatment of steatosis of a liver graft.

2. Composition according to claim 1, wherein the composition is in the form of an injectable solution.

3. A composition according to claim 1 or 2, wherein the composition is used in an ex live to the liver graft.

4. Composition according to claim 3, in which the administration ex live is a perfusion in normothermic conditions.

5. Composition according to claim 3 or 4, wherein the concentration of rapamycin in the composition is less than or equal to 1 mg / mL, preferably between 0.4 mg / mL and 1 mg / mL, preferably 0.5 mg / mL.

6. Composition according to any one of claims 3 to 5, in which the circulating concentration of rapamycin is between 120 ng / mL and 360 ng / mL, preferably 180 ng / mL.

7. Composition according to any one of claims 3 to 6, comprising between 70% and 99% of dimethylsulfoxide and between 1% and 30% of water, preferably 85% of dimethylsulfoxide and 15% of water, expressed as a volume percentage.

8. Composition according to claim 7, in which the dimethylsulfoxide / water ratio is between 2.5 and 95, preferably between 2.65 and 91.

25.

9. Composition according to any one of claims 1 to 8, in which the steatosis of the liver graft is greater than 30%.

10. Composition according to any one of claims 1 to 9, wherein the composition is stored between -30°C and -10°C, preferably at -20°C.

11. A composition according to claim 1 or 2, wherein the composition is used in an administration in vivo to the liver graft.

12. Composition according to any one of claims 1 to 11, in which the composition is obtainable by a preparation process comprising the following steps: 1) Dissolving rapamycin with dimethylsulfoxide at a temperature above 18°C; 2) Adding water at a temperature above 18°C ​​to the solution of rapamycin and dimethylsulfoxide obtained in step 1; 3) Sterilizing the solution obtained in step 2, preferably by filtration.

13. Composition according to claim 12, in which the rapamycin is dissolved in step 1 at a rate of 5 mg / mL.

14. Composition according to claim 12 or 13, in which the filtration is carried out with a nylon or polytetrafluoroethylene filter.

15. Method for administering a composition as defined in any one of claims 1 to 10 and 12 to 14, comprising the following steps: 1) Installation of a liver graft on a perfusion machine in hypothermic condition, preferably between 4°C and 8°C for 1 to 2 hours, then in normothermic condition, preferably between 36°C and 38°C for 12 to 120 hours; 2) Injection into the perfusion circuit of the composition comprising rapamycin, dimethyl sulfoxide and water, preferably 2 hours after the start of the normothermic condition.

16. Use of a composition comprising rapamycin, dimethyl sulfoxide and water for treating steatosis of a liver graft.

17. Use of a composition according to claim 16, wherein the composition is administered ex live to the liver graft.

18. Use of a composition according to claim 17, in which the concentration of rapamycin in the composition is less than or equal to 1 mg / mL, preferably between 0.4 mg / mL and 1 mg / mL, even more preferably 0.5 mg / mL.

19. Use of a composition according to claim 17 or 18, in which the circulating concentration of rapamycin is between 120 ng / mL and 360 ng / mL, preferably 180 ng / mL.

20. Use of a composition according to any one of claims 16 to 19, in which the composition comprises between 70% and 99% of dimethylsulfoxide and between 1% and 30% of water, preferably 85% of dimethylsulfoxide and 15% of water, expressed as a volume percentage.

21. Use of a composition according to any one of claims 16 to 20, in which the steatosis of the liver graft is greater than 30%.

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